Continuing the banner day, Daniel G. Gibson and colleagues have published a rather subdued article in Science. In this truly impressive experiment, a group from the J. Craig Venter Institute synthesized and assembled an entire 583 kilobase-pair genome of Mycoplasma genitalium(1). Besides being a fairly cool piece of work all on its own, this outcome has profound implications for the future of bacterial research and the development of custom organisms

The stated purpose of the research is to investigate M. genitalium itself, which has the convenient property of possessing a very small genome. As the authors note (emphasis mine):

Approximately 100 of its 485 protein-coding genes are nonessential… it is not known which of these 100 genes are simultaneously dispensable. We proposed that one approach to this question would be to produce reduced genomes by chemical synthesis…

One might reasonably conclude from this passage that JCVI has a dearth of small thinkers: this is akin to proposing to light your yard by shifting the position of the moon.

Yet Gibson et al. pulled it off using a tiered assembly method. They synthesized 101 overlapping pieces of DNA ~6 kb long. Using in vitro recombination they assembled groups of these into 24 kb fragments and propagated them using bacterial artificial chromosomes. Using a similar strategy they managed to continue assembling fragments up to about 144 kb in size; beyond this, however, the in vitro method did not work. So they turned to the yeast Saccharomyces cerevisiae to assemble the larger “C” fragments into half-genome and even whole-genome constructs. They then checked the whole genome for errors using the famous “shotgun” approach. Amazingly, their first run produced only two errors, both human in origin. After some wrangling they ironed it all out and ended up with precisely the genome they wanted, complete with special “watermarks”.

So they managed to construct an enormous hunk of DNA. What’s the benefit? Well, they can do those crazy experiments that they’re planning, for one thing. For another, assuming they can figure out how a mycoplasma works, they can design a new mycoplasma that does something special. For instance, they could design a new genome containing genes that allow the bacterium to eat toxic materials, or purify uranium out of the soil, or turn biomass into octane.

Granted, it is possible to get some of these genes into a bacterium using plasmids or BACs. However, what goes in can also go out—ejecting a plasmid is relatively easy, especially if it doesn’t provide any selective advantage. Completely ejecting a genomic cassette, while possible, is much more rare. Moreover, if you can custom-design the genome, then the custom enzymes can be interspersed with essential genes, making deletion that much more unlikely. Thus, once the genome can be manipulated at will, the only limitation on what an organism can be made to do is the extent of our ability to find or design proteins that fulfill the desired function. Figuring out which of a hundred-odd genes from M. genitalium can be done away with is just an efficiency step to make space for new genes to do whatever we want.

This approach will be a powerful tool for other experiments as well, don’t get me wrong. For instance, you could use the approach to place custom tags around certain genes so that they get excised at particular times or in a conditional manner, or you could swap sequences around to get a better idea how important gene arrangement is. But there’s no denying the enormous advance that this success makes possible, despite the authors’ decision not to speculate in the paper. The genome-synthesis technique is an essential step in the construction of a completely custom organism.